Cytochrome P450 3A4-catalyzed testosterone 6β-hydroxylation stereochemistry, kinetic deuterium isotope effects, and rate-limiting steps

Testosterone 6 beta-hydroxylation is a prototypic reaction of cytochrome P450 ( P450) 3A4, the major human P450. Biomimetic reactions produced a variety of testosterone oxidation products with 6 beta-hydroxylation being only a minor reaction, indicating that P450 3A4 has considerable control over the course of steroid hydroxylation because 6 beta-hydroxylation is not dominant in a thermodynamically controlled oxidation of the substrate. Several isotopically labeled testosterone substrates were prepared and used to probe the catalytic mechanism of P450 3A4: (i) 2,2,4,6,6-H-2(5); (ii) 6,6(2)H(2); (iii) 6 alpha-H-2; (iv) 6 beta-H-2; and (v) 6 beta-H-3 testosterone. Only the 6 beta-hydrogen was removed by P450 3A4 and not the 6 alpha, indicating that P450 3A4 abstracts hydrogen and rebounds oxygen only at the beta face. Analysis of the rates of hydroxylation of 6 beta-H-1-, 6 beta-H-2-, and 6 beta-H-3-labeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect ((D)k) of 15. The deuterium isotope effects on k(cat) and k(cat)/K-m in non-competitive reactions were only 2-3. Some "switching" to other hydroxylations occurred because of 6 beta-H-2 substitution. The high Dk value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high Dk in the non-competitive experiments implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high Dk value was also seen with a slower P450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking.